Unitary biochip providing sample-in to results-out processing and methods of manufacture

Abstract

A biochip for the integration of all steps in a complex process from the insertion of a sample to the generation of a result, performed without operator intervention includes microfluidic and macrofluidic features that are acted on by instrument subsystems in a series of scripted processing steps. Methods for fabricating these complex biochips of high feature density by injection molding are also provided.

What is claimed is: 
     
       1. A biochip for conducting a multiplexed PCR analysis of a at least two nucleic acids, said biochip to be used with an instrument, said instrument comprising a process controller and a pneumatic subsystem, said biochip comprising:
 (i) a pneumatic subassembly comprising at least one pneumatic subassembly interface to said pneumatic subsystem on said instrument and to the subassemblies of said biochip, comprising a pneumatic plate comprising one or a plurality of drive lines that contain only air said pneumatic plate aligned with a fluidics assembly via at least one through hole for each of said at least one preloaded elution reagent storage chambers on or in a macrofluidic block, and configured to receive pressure from said instrument and deliver it to said top ends thereof, thereby causing said first and second foils to burst, and to release the contents of each of said at least one preloaded reagent storage chambers upon scripted controls from said process controller; 
 (ii) said macrofluidic block comprising: a) at least one preloaded elution reagent storage chamber, each one of said preloaded elution reagent storage chambers having a top end and a bottom end, a first foil seal bonded to the bottom end, and a second foil seal bonded to the top end; and (b) an inlet for receiving said at least two nucleic acids; 
 (iii) said fluidics subassembly comprising: a) at least one reconstitution chamber preloaded with lyophilized multiplexed PCR reagent and positioned in fluid communication with said elution reagent storage chamber; b) at least one thermal cycling chamber; and (c) a primary flow path, said path in fluidic communication with said at least one reconstitution chamber and said at least one thermal cycling chamber, such that when said at least one preloaded elution reagent storage chamber receives pressure delivered to said top end from said instrument, said second foil bursts and releases the contents of each of said at least one elution reagent storage chambers, resulting in elution of said at least two nucleic acids followed by reconstitution of said lyophilized multiplexed PCR reagent, thereby generating PCR mix solution for amplification, whereupon said PCR mix solution travels along said flow path to said at least one thermal cycling chamber. 
 
     
     
       2. The biochip of  claim 1  wherein said lyophilized multiplexed PCR reagent contains at least 7 primer pairs. 
     
     
       3. The biochip of  claim 1  wherein said lyophilized multiplexed PCR reagent contains at least 16 primer pairs. 
     
     
       4. The biochip of  claim 1  wherein said lyophilized multiplexed PCR reagent contains at least 80 primer pairs. 
     
     
       5. The biochip of  claim 1  having at least one secondary flow path, said secondary flow path in fluidic communication with at least one second chamber, said at least one second chamber adapted for nucleic acid extraction, cell lysis, cell separation, differential cell lysis, differential filtration, total nucleic acid purification, DNA purification, RNA purification, mRNA purification, protein purification, pre-nucleic acid amplification cleanup, single nucleotide polymorphism analysis, VNTR analysis, RFLP analysis, post-nucleic acid amplification cleanup, pre-nucleic acid sequencing cleanup, Sanger sequencing, pyrosequencing; single molecule sequencing, post-nucleic acid sequencing cleanup, reverse transcription, pre-reverse transcription cleanup, post-reverse transcription cleanup, nucleic acid ligation, nucleic acid hybridization, electrophoretic separation and detection, immunoassay, binding assay, protein assay, enzymatic assay, mass spectroscopy, nucleic acid quantification or protein quantification. 
     
     
       6. The biochip of  claim 1  wherein said lyophilized multiplexed PCR reagent contains at least 7 primer pairs. 
     
     
       7. The biochip of  claim 6  wherein the lyophilized multiplex PCR reagent containing at least 7 primer pairs is also configured to perform Real-time PCR, reverse transcription PCR, asymmetric PCR, nested PCR, LATE PCR, touchdown PCR, digital PCR, rolling circle amplification, strand displacement amplification, multiple displacement amplification, STR amplification, Y-STR amplification or mini-STR amplification. 
     
     
       8. The biochip of  claim 1  wherein said lyophilized multiplexed PCR reagent contains at least 16 primer pairs. 
     
     
       9. The biochip of  claim 8  in which said at least 16 primer pairs are primer pairs for human STR loci. 
     
     
       10. The biochip of  claim 1 , comprising a second reconstitution chamber, said second reconstitution chamber preloaded with lyophilized sequencing reagent, and positioned on said primary flow path downstream from said at least one thermal cycling chamber. 
     
     
       11. The biochip of  claim 1  wherein a pressure of 20-500 psig is applied at the interface to burst at least one of the foils. 
     
     
       12. The biochip of  claim 1  wherein at least one of the foils is scored. 
     
     
       13. The biochip of  claim 1  wherein the lyophilized multiplexed PCR reagent further comprises a lyophilized reverse transcriptase and the reconstituted PCR/reverse transcriptase solution is subjected to reverse transcription followed by amplification in said thermal cycling chamber. 
     
     
       14. A unitary biochip for conducting a multiplexed PCR analysis of at least two nucleic acids, said biochip to be used with an instrument, said instrument comprising a process controller and a pneumatic subsystem, said biochip comprising:
 (i) a pneumatic subassembly comprising at least one pneumatic subsystem interface to said pneumatic subsystem on said instrument and to the subassemblies of said biochip, comprising a pneumatic plate comprising one or a plurality of drive lines that contain only air, said pneumatic plate aligned with a fluidics subassembly via at least one through hole for each of the at least one preloaded elution reagent storage chambers on or in a macrofluidic block, and configured to receive pressure from said instrument and deliver it to said top ends thereof, thereby causing said first and second foils to burst, and to release the contents of each of said at least one preloaded reagent storage chambers upon scripted controls from said process controller; 
 (ii) said macrofluidic block comprising:
 a) said at least one preloaded elution reagent storage chamber, each one of said preloaded elution reagent storage chambers having a top end and a bottom end, a first foil seal bonded to the bottom end, and a second foil seal bonded to the top end; and 
 b) an inlet for receiving said at least two nucleic acids; 
 
 (iii) said fluidics subassembly comprising:
 a) at least one combination reconstitution/thermal cycling chamber preloaded with lyophilized multiplexed PCR reagent, and positioned in fluid communication with said elution regent storage chamber, 
 b) a primary flow path, said path in fluidic communication with said microfluidic block at said least one combination reconstitution/thermal cycling chamber; 
 
 such that when said preloaded elution reagent storage chamber receives pressure delivered to said top end from said instrument, said second foil bursts and releases the contents of the elution reagent storage chamber, resulting in elution of said at least two nucleic acids followed by reconstitution of said lyophilized multiplexed PCR reagent, thereby generating PCR mix solution for amplification, followed by direct amplification of said PCR mix solution in said combination reconstitution/thermal cycling chamber. 
 
     
     
       15. A unitary biochip for conducting a multiplexed PCR analysis of at least five nucleic acid solutions, said biochip to be used with an instrument, said instrument comprising a process controller and a pneumatic subsystem, said biochip comprising:
 (i) a pneumatic subassembly comprising at least one pneumatic subsystem interface to said instrument and to the subassemblies of said biochip, comprising a pneumatic plate comprising one or a plurality of drive lines that contain only air said pneumatic plate aligned with a fluidics assembly via at least one through hole for each of said at least one preloaded elution reagent storage chamber on or in a macrofluidic block, and configured to receive pressure from said instrument and deliver it to said top ends thereof, thereby causing said first and second foils to burst, and to release the contents of each of said at least one preloaded reagent storage chambers upon scripted controls from said process controller; 
 (ii) said macrofluidic block comprising:
 a) at least five preloaded elution reagent storage chambers, one preloaded elution reagent storage chamber for each of said at least five nucleic acid solutions, each one of said preloaded elution reagent storage chambers having a top end and a bottom end, a first foil seal bonded to the bottom end, and a second foil seal bonded to the top end; and 
 b) an inlet for receiving said at least two nucleic acids; 
 
 (iii) said fluidics subassembly comprising:
 a) at least five reconstitution chambers, one reconstitution chamber for each of said nucleic acid solutions, each of said five reconstitution chambers preloaded with lyophilized multiplexed PCR reagent, and positioned in fluid communication with said elution regent storage chambers, 
 b) at least five thermal cycling chambers one thermal cycling chamber for each of said nucleic acid solutions; and 
 c) at least five primary flow paths, each of said five paths in fluidic communication with said at least one reconstitution chamber and said at least one thermal cycling chamber; 
 
 such that when each of said preloaded elution reagent storage chambers receives pressure delivered to said top end from said instrument, said second foil bursts and releases the contents of the elution reagent storage chamber, resulting in elution of nucleic acids in said at least five nucleic acid solutions, followed by reconstitution of said lyophilized multiplexed PCR reagent, thereby generating PCR mix solution for amplification, whereupon said PCR mix solution travels along said flow path to each of said at least five thermal cycling chambers. 
 
     
     
       16. The biochip of  claim 14  having at least one secondary flow path, said secondary flow path in fluidic communication with at least one second chamber,
 said at least one second chamber adapted for nucleic acid extraction, cell lysis, cell separation, differential cell lysis, differential filtration, total nucleic acid purification, DNA purification, RNA purification, mRNA purification, protein purification, pre-nucleic acid amplification cleanup, single nucleotide polymorphism analysis, VNTR analysis, RFLP analysis, post-nucleic acid amplification cleanup, pre-nucleic acid sequencing cleanup, Sanger sequencing, pyrosequencing, single molecule sequencing, post-nucleic acid sequencing cleanup, reverse transcription, prereverse transcription cleanup, post-reverse transcription cleanup, nucleic acid ligation, nucleic acid hybridization, electrophoretic separation and detection, immunoassay, binding assay, protein assay, enzymatic assay, mass spectroscopy, nucleic acid quantification or protein quantification, 
 further comprising at least one additional fluid transport channel and a chamber adapted for at least one of the group consisting of nucleic acid extraction, cell lysis, cell separation, differential cell lysis, differential filtration, total nucleic acid purification, DNA purification, RNA purification, mRNA purification, protein purification, pre-nucleic acid amplification cleanup, single nucleotide polymorphism analysis, VNTR analysis, RFLP analysis, post-nucleic acid amplification cleanup, pre-nucleic acid sequencing cleanup, Sanger sequencing, pyrosequencing and single molecule sequencing, post-nucleic acid sequencing cleanup, reverse transcription, pre-reverse transcription cleanup, post-reverse transcription cleanup, nucleic acid ligation, nucleic acid hybridization, electrophoretic separation and detection, immunoassay, binding assay, protein assay, enzymatic assay, mass spectroscopy, nucleic acid quantification and protein quantification.